1. Field of the Invention
This invention relates to the genetic manipulation of plants of the family Cruciferae (Brassicaceae) to improve the quality and usefulness of seed meal obtained from such plants. More particularly, the invention relates to such genetic manipulation of species of Cruciferae to decrease the content of sinapine in the seeds and seed meal thereof.
2. Background Art
Several species of the family Cruciferae are grown on a commercial scale for the production of oils from the plants seeds. Rape (Brassica rapa; Brassica napus) and Canola (Brassica napus; Brassica rapa) are examples of such species, among others. The oils extracted from the plant seeds have various uses, e.g. as cooking oils, lubricants and consumable foodstuffs, but the residues of the seeds after oil extraction (referred to as seed meal) also have commercial uses. Canola meal, for example, which forms about 60% of the seed weight and contains about 36-44% crude protein, is primarily used as a protein supplement in animal feeds. However, the use of such seed meals in animal diets is somewhat limited due to presence of several anti-nutritional factors. Among them, sinapine, a major sinapate-derived phenolic ester present in the seeds, imparts a bitter taste to the meal and makes it less palatable to animals (Bell, 1993). The presence of sinapine in the meal also limits its use as a feed supplement for poultry diet as it results in eggs with fishy taint (Bell, 1993).
Sinapine is a metabolic product that accumulates in such seeds supposedly as a reserve seed material and that remains in the solid seed meal after oil extraction. For example, sinapine constitutes 1-4% of dried canola meal. During seed germination, sinapine is hydrolysed (Strack, 1981); however, it is not clear whether sinapine is essential for seed germination and seedling development. Recent identification of an Arabidopsis mutant that fails to accumulate sinapine in seeds but still undergoes normal seed germination, suggests that sinapine is dispensable for seed germination and seedling growth (Chapple et al., 1992). Efforts to develop oil seed (e.g. B. napus) breeding lines with low sinapine contents have not been successful, so it would be desirable to produce low-sinapine variants of oilseed species of Cruciferae by other means.
An object of the invention is to genetically transform plants of the family Cruciferae (Brassicacea), for example B. napus, to make the seeds, and particularly the seed meal, thereof more useful and commercially valuable.
Another object of the invention is to reduce the sinapine content of seeds and seed meal of Crucifera plants compared to wild type or vector control plants of the same species.
According to one aspect of the present invention, there is provided a transformed seed-producing plant, or a part thereof, of the Crucifera family containing an exogenous DNA sequence operably linked to a plant promoter for expression of a polypeptide that acts as a CYP84 monooxygenase enzyme, or a polypeptide that is an antisense equivalent of an CYP84 monooxygenase enzyme, or a polypeptide that is a catalytic fragment or derivative of said monooxygenase enzyme or antisense equivalent that does not alter the function of said CYP84 enzyme or antisense equivalent, and which plant or plant part has a reduced content of sinapine in seeds thereof compared to vector control plants or plant parts of the same species lacking said exogenous DNA sequence.
According to another aspect of the invention, there is provided a method of producing a transformed seed-producing plant, or part thereof, of the Crucifera family having a reduced content of sinapine in seeds thereof compared to vector control plants of the same species lacking said exogenous DNA sequence, said method comprising transforming cells of said plant or plant part with an exogenous DNA sequence operably linked to a plant promoter for expression in said plant cell of a polypeptide that acts as a CYP84 monooxygenase enzyme, or a polypeptide that is an antisense equivalent of an CYP84 monooxygenase enzyme, or a polypeptide that is a catalytic fragment or derivative of said monooxygenase enzyme or antisense equivalent, and selecting transformed plants or plant parts having reduced content of sinapine in seeds thereof compared to vector control plants or plant parts of the same species lacking said exogenous DNA sequence.
According to another aspect of the invention, there is provided a seed meal prepared by growing the plant or part thereof as described above, harvesting seeds from the plant, and processing said seeds to form seed meal.
According to another aspect of the invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or an active fragment, derivative, homolog or analog thereof, or a sequence that is complementary to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or an active fragment thereof, derivative, homolog or analog thereof., or a sequence having at least 90% identity to said sequence or complementary sequence.
According to yet another aspect of the invention, there is provided a chimeric gene causing reduced sinapine content in plant cells of the Crucifera family transformed with the chimeric gene, the chimeric gene comprising: a regulatory nucleotide sequence; and a nucleic acid fragment encoding an active plant CYP84 enzyme; and wherein the nucleic acid fragment is operably linked in either the sense or antisense orientation to the regulatory sequence.
According to yet another aspect of the invention, there is provided a seed meal comprising seeds, or parts thereof, of a transformed seed-producing plant of the Crucifera family containing an exogenous DNA sequence operably linked to a plant promoter for expression of a polypeptide that acts as a CYP84 monooxygenase enzyme, or a polypeptide that is an antisense equivalent of an CYP84 monooxygenase enzyme, or a polypeptide that is a catalytic fragment or derivative of said monooxygenase enzyme or antisense equivalent that does not alter the function of said CYP84 enzyme or antisense equivalent, and which plant or plant part has a reduced content of sinapine in seeds thereof compared to vector control plants or plant parts of the same species lacking said exogenous DNA sequence.
According to a still further aspect of the invention, there is provided a method of producing a transformed seed-producing plant, or part thereof, of the Crucifera family having a reduced content of sinapine in seeds thereof compared to vector control plants of the same species lacking said exogenous DNA sequence, said method comprising transforming cells of said plant or plant part with an exogenous DNA sequence operably linked to a plant promoter for expression in said plant cell of a polypeptide, said DNA sequence being a sequence flanking an endogenous CYP84 gene of the plant, or a sequence that is complementary thereto.
The sequence of the invention preferably expresses an antisense equivalent of a CYP84 monooxygenase or a catalytic fragment or derivative thereof, and the CYP84 monooxygenase is preferably a enzyme capable of hydroxylating coniferaldehyde or ferulate or coniferyl alcohol or other substrates, most preferably derived from B. napus. 
The invention also relates to vectors used in the transformation of the plants, and polypeptide sequences produced by expression of the indicated DNA sequences.
The ferulate 5-hyrdoxylase enzyme is currently believed capable of hydroxylating primarily coniferaldehyde and to a lesser extent coniferyl alcohol or ferulic acid or a precursor leading to sinapine. Preferably, the polypeptide what has been referred variously as ferulic acid hydroxylase, ferulate 5-hydroxylase, coniferaldehyde 5-hydroxylase or abbreviated as FAH, F5H, CAld5H or also as CYP84 family of P450 dependent monooxygenase.
The invention also relates to an antibody molecule which binds to the polypeptide of the invention, vectors containing genetic constructs according to the invention, a transgenic Brassica napus plant transformed with the isolated nucleic acid molecule, a transgenic Brassica napus plant transformed with the genetic construct defined above, a seed of Brassica napus transformed with the isolated nucleic acid molecule or the genetic construct and the use of an isolated nucleic acid molecule to reduce the sinapine content of seeds of Brassica napus, and canola meal made from the transformed Brassica napus seeds.
The invention has the advantage that the transformed plants, and progeny thereof, have the ability to produce seeds having a reduced content of sinapine that produces a more valuable and useful seed meal by-product.